The methylation status of DNA influences many biological processes during mammalian development, and is known to be highly aberrant in cancer. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 1-ketoglutarate (1KG)- and Fe(II)-dependent dioxygenases that alter DNA methylation status by catalysing the oxidation of 5-methylcytosine (5mC) to 5-hydroxy- methylcytosine (5hmC) in DNA. Tet1, Tet2 and 5hmC are present at high levels in mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, suggesting a potential role for 5hmC in pluripotency. Moreover, 5hmC levels and Tet expression/ activity are tightly regulated: 5hmC is present in genomic DNA of undifferentiated ES cells but not several differentiated cell types, and 5hmC levels diminish upon ES cell differentiation. Together these data suggest that dysregulation of DNA methylation via TET proteins and 5hmC may have a role in ES cell differentiation and function. Here we propose to analyze the biological roles of Tet proteins in gene expression, pluripotency and cell fate specification in mouse ES and iPS cells. In Aim 1, we will study the roles of Tet proteins in mouse ES cell differentiation and function in vitro and in vivo using Tet-deleted ES cells and mice. In Aim 2, we will examine the requirement for Tet proteins in reprogramming murine fibroblasts to iPS cells. In Aim 3, we will identify target genes for Tet proteins in ES cells by transcriptional profiling, defining the genomic locations of 5hmC and Tet proteins, and mapping the location of 5hmC at single- base resolution in the genome. The results should provide new insights into the role of the novel base, 5hmC, and the newly-discovered TET family of enzymes, in pluripotency and stem cell function in ES and iPS cells. PUBLIC HEALTH RELEVANCE: Role of TET proteins in ES cell pluripotency and function Narrative In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a very minor base known as 5-methylcytosine (5mC) that has a disproportionately crucial role. This base is produced from the major base cytosine (C) by attaching a methyl group to its "5" position. Interference with cytosine methylation can lead to a number of developmental abnormalities, genetic diseases and cancer. We recently identified a new class of proteins known as TET proteins that convert 5-methyl- cytosine to a variant known as 5-hydroxymethylcytosine (5hmC). TET proteins and 5-hmC are strongly expressed in mouse embryonic stem (ES) cells, and are induced to high levels when mouse fibroblasts are reprogrammed into induced pluripotent stem (iPS) cells. In this proposal we plan to investigate the role of TET1 and TET2 proteins in ES and iPS cells.